Role of endothelial nitric oxide synthase for early brain injury after subarachnoid hemorrhage in mice

Abstract

The first few hours and days after subarachnoid hemorrhage (SAH) are characterized by cerebral ischemia, spasms of pial arterioles, and a significant reduction of cerebral microperfusion, however, the mechanisms of this early microcirculatory dysfunction are still unknown. Endothelial nitric oxide production is reduced after SAH and exogenous application of NO reduces post-hemorrhagic microvasospasm. Therefore, we hypothesize that the endothelial NO-synthase (eNOS) may be involved in the formation of microvasospasms, microcirculatory dysfunction, and unfavorable outcome after SAH. SAH was induced in male eNOS deficient (eNOS^-/-) mice by endovascular MCA perforation. Three hours later, the cerebral microcirculation was visualized using in vivo 2-photon-microscopy. eNOS^-/- mice had more severe SAHs, more severe ischemia, three time more rebleedings, and a massively increased mortality (50 vs. 0%) as compared to wild type (WT) littermate controls. Three hours after SAH eNOS^-/- mice had fewer perfused microvessels and 40% more microvasospasms than WT mice. The current study indicates that a proper function of eNOS plays a key role for a favorable outcome after SAH and helps to explain why patients suffering from hypertension or other conditions associated with impaired eNOS function, have a higher risk of unfavorable outcome after SAH.

Keywords: Subarachnoid hemorrhage; early brain injury; endothelial NOS; microvasospasm; nitric oxide.

Figure 1.

Intravital microscopy - Experimental setup. (a) Schematic overview of cranial window placement for intravital microscopy. (b) Assessment of microvasospams formation and severity. (c) Example of a hematoma at the skull base after SAH (left) and the quantification of hematoma size (green) by an automated algorithm (right).

Figure 2.

Intracranial pressure and local cerebral blood following subarachnoid hemorrhage. (a) Peak intracranial pressure is significantly and gene-dose-dependently elevated in eNOS deficient mice. After few minutes, ICP drops again but remains significantly elevated in eNOS homozygous animals compared to wild type littermates. (b) Cerebral perfusion drops to lower values in eNOS deficient as compared to wild type mice. While in heterozygous and wild type mice CBF recovers to baseline values within a short time, CBF in homozygous eNOS deficient mice remains significantly impaired.

Figure 3.

Re-bleeding events. (a) Exemplary ICP traces in single mice show repetitive re-bleedings in an eNOS^–/– animal while no such events were observed in a wild type littermate. (b) Quantification reveals a significantly elevated number of re-bleeding events in eNOS homozygous animals compared to wild type and heterozygous mice (p = 0.004 and 0.022, respectively). (c) Invasive arterial blood pressure recordings in homozygous (dark grey symbols) and heterozygous (light grey symbols) eNOS deficient mice and wild type littermates before and after SAH. Already before SAH eNOS^–/– mice had a slightly increased arterial blood pressure. Within the first four minutes after SAH all three genotypes showed the typical increase in blood pressure due to increased intracranial pressure, the Cushing response. Thereafter, arterial blood pressure returned to baseline levels in all investigated genotypes.

Figure 4.

Possible causes for increased SAH severity in eNOS^–/– mice. (a) eNOS^–/– mice had significantly larger hematomas at the skull base as compared to wild type littermates after SAH. (b) Tail bleeding time was significantly elevated in eNOS deficient animals.

Figure 5.

Changes of the cerebral microcirculation after SAH. (a) Exemplary IVM screenshots depicting the cerebral microcirculation in naïve animals (upper panels) and after induction of SAH (lower panels). eNOS deficient mice already show a slight narrowing and rarefication on the capillary level. After SAH, there is (increased) microvascular constriction in both genotypes. (b) Corresponding to the exemplary picture, total perfused vessel volume is slightly but significantly lower in eNOS^–/– homozygous animals compared to wild type mice. After SAH, perfused vessel volume is significantly lower in both genotypes, indicating relevant microvascular constriction and – thus – microcirculatory dysfunction. The drop in perfused vessel volume, however, is significantly more pronounced in eNOS^–/– than in wt. (c) Microvasospasms occur very rarely under physiological conditions in wild type controls (left side, dark bar). The number of MVS in eNOS^–/– is significantly elevated. After SAH, there is significant spasm formation in wt mice (right side of panel). This increase is more pronounced in eNOS^–/– mice (striped bars).

Figure 6.

Mortality. While all wild type animals survived the early phase after SAH and heterozygous animals showed a moderately elevated mortality rate of 25%, nearly half of all homozygous eNOS^–/– mice died within the first 3 hours after hemorrhage induction.